In general, the concentration of cholinesterase in serum is known to be decreased, for example, in a patient with liver disease, while it is known to be increased, for example, in a patient with kidney disease. Therefore, these diseases can be diagnosed by determining the cholinesterase activity in serum of these patients and a determination method which permits exact determination of cholinesterase activity in serum can be used for clinical examinations.
As methods for determining cholinesterase (hereinafter referred to as ChE) activity in serum, there have heretofore been reported various methods using a synthetic substrate, and some of them have been made practicable for daily clinical examinations. However, these determination methods involve various defects and problems, and these disadvantages are responsible for the inaccuracy of the resulting determined value. Examples of the heretofore well-known determination methods include (a) gas analysis method, (b) pH meter method, (c) pH-indicator colorimetric method, (d) thiocholine color formation method, (e) enzymatic method, (f) UV method, etc.
(a) The gas analysis method [R. Ammon: Pflugers Arch. Ges Physiol., 233, 487 (1933)] comprises using acetylcholine as a synthetic substrate, bringing acetic acid produced by the enzymatic action of ChE into contact with sodium hydrogen carbonate, and quantitatively determining the carbon dioxide gas produced. This method, however, is disadvantageous, for example, in that since its operations are troublesome, it cannot deal with many samples.
(b) The pH meter method [H. O. Michel: J. Lab. & Clin. Med., 34, 1564 (1949)], like the gas analysis method, comprises using acetylcholine as a synthetic substrate, and measuring a pH change due to acetic acid produced by the enzymatic action of ChE by means of a pH meter. This method, however, involves a problem of low accuracy of pH meter and involves practical problems of, for example, inability to deal with many samples, and the like.
(c) The pH-indicator colorimetric method, unlike the pH meter method, comprises using acetylcholine as a synthetic substrate, and measuring a pH change due to acetic acid produced by ChE in terms of the molecular absorbance of the indicator. As the indicator, there are used phenol red [Hiroshi Takahashi and Susumu Shibata, IGAKU-TO-SEIBUTSUGAKU (Medicine and Biology), 20, 96, (1959)], bromothymol blue [H. G. Biggs, et al., Amer. J. Clin. Path., 30, 181, (1958)], m-nitrophenol [Tadahide Sasaki, RINSHO-BYORI (Clinical Pathology), 12, 555, (1964)], etc. This method comprises simple operations and can deal with many samples, but it is pointed out that this method is disadvantageous, for example, in that the reaction time is long and in that during the reaction, the pH is not constant and is not sufficiently reproducible at low and high values.
All of the above-mentioned methods (a) to (c) use acetylcholine as a substrate, and in the case of such methods, the substrate itself also is disadvantageous because acetylcholine tends to undergo nonenzymatic hydrolysis and has no sufficient substrate specificity.
(d) The thiocholine method [P. Garry, J. Clin. Chem., 11 (2), 91 (1965)] uses acetylthiocholine, propylthiocholine, butylthiocholine or the like as a substrate. These substrates yields thiocholine by the enzymatic reaction of ChE, and this thiocholine reacts with 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) to produce a yellow color. The thiocholine method comprises measuring this yellow color by means of a colorimeter. This method is advantageous, for example, in that it is excellent in reactivity, has a high sensitivity, comprises simple operations, can deal with many samples, and makes it possible to carry out the determination also by an initial velocity method. However, it is seriously affected by bilirubin in serum because of the yellow coloration and unavoidably affected by compounds having a thiol group such as glutathione. Furthermore, it is disadvantageous, for example, in that the instability of the substrate itself is a problem. These disadvantages are responsible for errors of determined values.
(e) The enzymatic method comprises using benzoylcholine [Hiroaki Okabe et al., RINSHO-BYORI (Clinical Pathology), 25, 751, (1977)], orthotoluoylcholine [Japanese Patent Application Kokai (Laid-Open) No. 138533/79] or the like as a substrate, converting choline produced by the enzymatic action of ChE into betaine by cholineoxidase, and subjecting 4-aminoantipyrine to oxidative condensation reaction with phenol or the like by thus produced hydrogen peroxide in the presence peroxidase to cause color production. In this method, since the coloration is red, there is not interference by bilirubin in serum, and many samples can be dealt with. However, since phenol or 4-aminoantipyrine used as a reagent for the color-producing system competitively inhibits ChE, the amount of these reagents used is greatly limited, so that sufficient color production is difficult. In general, a determination method via hydrogen peroxide is unavoidably affected not only by bilirubin in serum, reducing substances such as ascorbic acid and the like, etc. but also by choline produced by decomposition of phospholipids or the like. In particular, the employment of benzoylcholine as a substrate involves various problems, for example, its nonenzymatic hydrolyzability which causes troubles.
(f) The UV method includes various kinds of methods, and one of them is a method of W. Kalow using benzoylcholine as a substrate [W. Kalow and K. Genet, Can. J. Biochem. & Physical., 35, 339 (1957)], while another is a method using p-hydroxybenzoyl choline [Japanese Patent Application Kokai (Laid-Open) Nos. 110198/82 and 129999/83] as a substrate. The former comprises following a decrease in amount of the substrate caused by its hydrolysis by the enzymatic action of ChE at a determination wave length of 240 nm, measuring the change in absorbance per definite time, and thereby determining ChE activity. The principle of determination of this method is simple and plain because the decrease of the substrate is directly determined. However, this method is disadvantageous, for example, in that since the determination wave length is 240 nm, interference by serum components tends to occur, that since benzoylcholine, i.e., the substrate causes substrate inhibition, the substrate concentration of the reaction solution is limited, resulting in a narrow range of linearity, and that since nonenzymatic hydrolysis of benzoylcholine tends to occur, the reaction can not be carried out in the optimum pH range of ChE. It is disadvantageous also, for example, in that since the determination wave length is 240 nm, the determination is carried out on the slope of absorption spectrum, resulting in a large deviation of absorption coefficient due to the deviation of wave length.
The latter comprises using p-phydroxybenzoylcholine as a substrate, reacting p-hydroxybenzoate hydroxylase with p-hydroxybenzoic acid produced by the enzymatic action of ChE, in the presence of the coenzyme NADPH, and determining and following, at a wave length of 340 nm, a decrease of absorbance at the time of oxidation of NADPH into NADP by this reaction. This method is an excellent method for determining ChE activity which makes it possible to carry out the reaction at an almost optimum pH, permits removal of the defects of the hydrogen peroxide color-producing system, namely, influence of bilirubin, reducing substances such as ascorbic acid and the like, etc. and interference by choline produced by decomposition of phospholipids, is free from the defects of the thiocholine method, and is suitable for an autoanalyzer capable of dealing with many samples. However, since NADPH, the coenzyme used, is an expensive reagent and is poor in stability, it is difficult to control while being kept at a definite quality. Further, in this method, p-hydroxybenzoate hydroxylase, protocatechuate 3,4-dioxygenase or the like is used as a reagent enzyme in the determination and moreover the principle of determination is considerably complicated as compared with the former determination method; therefore there are many factors which produce an error of the resulting determined value.
Further another example of the UV method is a method using 3,4-dihydroxybenzoylcholine iodide as a synthetic substrate (European Patent Publication No. 0160980). This method is a very excellent method for determining ChE activity which makes it possible to carry out the reaction in the optimum pH range of ChE, and is free from interference by other components in serum. In this method, the rate of the decrease of the substrate due to hydrolysis of said substrate by enzyme action of ChE is measured at a wave length of 340 to 360 nm, and in this wave length range, the measuring of the absorbance is carried out at the portion of the slope of absorption spectrum, so that an error of the resulting determined value tends to be produced depending on the kind of measuring instrument, etc. Therefore, this method admits of some improvement in the interchangeability of apparatus when the determination is carried out by means of an ordinary apparatus selected in a wide range.
On the other hand, as cholinesterases, there are known two kinds, namely, pseudo-cholinesterase existing in serum and true-cholinesterase existing in erythrocyte. Further, the pseudo-cholinesterase includes two kinds thereof, i.e., abnormal pseudo-cholinesterase and normal pseudo-cholinesterase. Cholinesterase whose activity is usually determined in a clinical examination is pseudo-cholinesterase in serum, but since serum is contaminated with true-cholinesterase in some cases, a substrate which reacts selectively with pseudo-cholinesterase alone is preferable as a synthesized substrate used in a method for determining ChE. As to a method for determining ChE activity, determination of abnormal pseudo-cholinesterase activity is also important. For determining abnormal pseudo-cholinesterase, there can be employed, for example, the above-mentioned UV method using p-hydroxybenzoylcholine. In this case, sodium fluoride is used in some cases in carrying out the determination. When sodium fluoride is used, the method using p-hydroxybenzoylcholine is seriously affected by sodium fluoride and hence is not suitable for determining abnormal pseudo-cholinesterase activity.
As described above, the conventional methods for determining the enzymatic activity of ChE involve various problems, and therefore it is desired to develop a method for determining ChE activity which has advantages such as no error in the determined value, good reproducibility of the determined value, and high substrate specificity for pseudo-cholinesterase.